|Year : 2013 | Volume
| Issue : 2 | Page : 135-141
Antieosinophilic activity of various subfractions of leaves of Vitex negundo
Jignesh I Patel1, Shrikalp S Deshpande2
1 Department of Pharmacology, B.K. Mody Government Pharmacy College, Polytechnic Campus, Near Aji-dam Chokdi, Rajkot- 360 003, Gujarat, India
2 Department of Pharmacology, K.B. Institute of Pharmaceutical Education and Research, Gandhinagar, Gujarat, India
|Date of Submission||19-Apr-2012|
|Date of Acceptance||15-May-2012|
|Date of Web Publication||3-Jun-2013|
Jignesh I Patel
Department of Pharmacology, B.K. Mody Government, Pharmacy College, Polytechnic Campus, Near Aji-dam, Chokdi, Rajkot - 360 003, Gujarat
Source of Support: K.B. Institute of Pharmaceutical Education and Research, Gandhinagar, Gujarat, India, Conflict of Interest: None
| Abstract|| |
Aim: The present study was undertaken to isolate and identify active constituent of leaves of Vitex negundo for their actions on bronchial hyperresponsiveness. Materials and Methods: Effects of aqueous subfraction, acetone subfraction, and chloroform sub-fraction on bronchial hyperresponsiveness and serum bicarbonate level were studied using egg-albumin-induced asthma in guinea pigs. The structure of aqueous subfraction of leaves of Vitex negundo was determined by various spectroscopy methods like UV spectroscopy, FT-IR spectroscopy, EI-mass spectroscopy, and H 1 NMR spectroscopy. Results: Animals pretreated with aqueous subfraction of leaves of Vitex negundo showed significantly lesser serum bicarbonate level, when compared with untreated sensitized animals. Animals pretreated with aqueous subfraction showed significant lesser eosinophils count (9.50 ± 1.5044; P < 0.05) when compared with untreated sensitized animals (29.50 ± 2.5074; P < 0.05). Histopathology of lungs in sensitized guinea pig showed infiltration of inflammatory cells like eosinophils and neutrophils which is due to marked inflammation in lungs. Histopathology of lungs in animals pretreated with aqueous subfraction (200 mg/ kg) showed normal airway, blood vessels, and bronchoalveolar space. Conclusion : It can be concluded that aqueous subfraction of leaves of Vitex negundo possesses antieosinophilic activity. It reduces bronchial hyperresponsiveness. As per UV, EI-Mass, FT-IR, and H 1 NMR spectra analysis, the structure of aqueous subfraction is determined as a 5-hydroxy-3,6,7,3',4'-pentamethoxy flavone. It may be useful in the treatment of asthma and various inflammatory, allergic, and immunologic diseases.
Keywords: 5-hydroxy-3,6,7,3′,4′-pentamethoxy flavones, eosinophils, spectroscopy methods, Vitex negundo
|How to cite this article:|
Patel JI, Deshpande SS. Antieosinophilic activity of various subfractions of leaves of Vitex negundo. Int J Nutr Pharmacol Neurol Dis 2013;3:135-41
|How to cite this URL:|
Patel JI, Deshpande SS. Antieosinophilic activity of various subfractions of leaves of Vitex negundo. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2015 May 24];3:135-41. Available from: http://www.ijnpnd.com/text.asp?2013/3/2/135/112839
| Introduction|| |
Eosinophils are thought to play a critical role in allergic diseases, such as allergic rhinitis, atopic dermatitis, and asthma. Eosinophils are present in peripheral blood, bronchial mucosa and bronchoalveolar lavage fluid of patients with asthma, and number of cells has been correlated with degree of hyperresponsiveness. Eosinophils are the source of lipid-derived mediators and eosinophils granules contain major basic protein (MBP), eosinophil cationic protein (ECP), eosinophils derived neurotoxin, and eosinophil peroxidase. Cytokines are also generated by eosinophils. MBP and ECP are detectable in sputum of asthmatic patients and may be responsible for damage to airway epithelium, which may in turn contribute to bronchial hyperresponsiveness(BHR).  Inflammation is the response of a tissue and its microcirculation to pathogenic injury. It is characterized by generation of inflammatory mediators and movement of fluid and leucocytes from the blood to extravascular tissue. 
A large number of drugs belonging to β2 agonists, corticosteroids, mast cell stabilizers, methylxanthines, leucotriene antagonists and others are in use for treating asthma. However none of them seems to be an ideal drug. The search for a new drug is still the need of the day. Herbal medicines are in great demand and are used by approximately 80% of the world's population. Their popularity is largely due to their presumed safety, efficacy, cultural acceptability, and lesser side effects compared with prescription medications.  There is high prevalence of usage of the alternative traditional system of medicines for the treatment of asthma. Ayurveda offers a unique insight into a comprehensive approach to asthma management through proper care of the respiratory tract. More than 400 medicinal plant species have been used ethnopharmacologically and traditionally to treat the symptoms of asthmatic and allergic disorders worldwide. Research on plants with medicinal properties and identification of the chemical components responsible for their activities have corroborated the traditional uses of ancient healing wisdom and lore and have proven the enduring healing potential of many plant medicines even in today's hi-tech community. The World Health Organization (WHO) has recognized herbal medicine as an essential building block for primary health care of vast countries like India and China. Herbal medicines are a treasure house of the information, from which we may derive leads to fill many blank spots in the modern medicine. 
Vitex negundo is a species reported under the genus Vitex. Family: Verbenaceae.  It is a large, aromatic shrub growing to a small tree. It commonly bears tri- or penta-foliate leaves on quadrangular branches, which give rise to bluish-purple colored flowers in branched tomatoes cymes. It thrives in humid places or along water courses in wastelands and mixed open forests and has been reported to occur in Afghanistan, India, Pakistan, Sri Lanka, Thailand, Malaysia, eastern Africa, and Madagascar. It is grown commercially as a crop in parts of Asia, Europe, North America, and the West Indies.  Vitex negundo seems to be a promising plant for treatment of bronchial asthma because of its reported immunomodulatory and anti-inflammatory activity. , Ethanolic extract of Vitex negundo leaves showed an inhibitory effect of degranulation of rat peritoneal mast cells induced by compound 48/80 and egg-albumin.  Aqueous extract of mature leaves of Vitex negundo showed an anti-inflammatory effect.  Leaf extract of V.negundo showed an anti-inflammatory effect on cotton pellet-induced granuloma in rats.  Ethyl acetate fraction of of Vitex negundo shows antiallergic, anti-eosinophilic, antioxidant, mast cell stabilizing activity of leaves. 
In the light of the above-mentioned facts, the objective of our present investigation was to evaluate the antieosinophilic activity of various subfraction of leaves of Vitex negundo and to isolate active constituent that possesses antieosinophilic activity and to identify the structure of active constituent by various spectroscopy methods.
| Materials and Methods|| |
The leaves of Vitex negundo plant were collected from Agriculture Research Centre, Jagudan, Mehsana, North Gujarat, India. The selected plant was authenticated by Dr. Vrinda Thaker, Professor, Department of Biosciences, Saurashtra University, Rajkot, Gujarat.
The protocol for this study was approved by institutional animal ethics committee (IAEC) of B.K. Mody Government Pharmacy College, Rajkot as per CPCSEA guidelines. IAEC approval no. was BKMGPC/IAEC08/RP01/2010. Healthy and fresh guinea pigs were procured from animal vaccine institute, Gandhinagar, Gujarat, India.
Preparation of plant fractions and sub-fractions
Leaves of Vitex negundo were dried under shade for a week. It was powdered and extracted with petroleum ether as a solvent to remove fatty material. Petroleum ether extract was devoid of antiasthmatic activity.  Residue was further extracted with ethyl acetate as a solvent using a soxhlet extractor since it was reported that ethyl acetate fraction possesses antiasthmatic activity. 
Ethyl acetate fraction of leaf (20 g) was further subfractionated twice with 2M sodium hydroxide through shaking and vortexing for 30 minutes. Sodium hydroxide filtrate was collected and liquid-liquid extraction with chloroform was done in separating funnel for 2 hours by shaking and the separating funnel was kept overnight for complete extraction. Upper layer containing sodium hydroxide portion was collected from separating funnel. 2N HCl was added in sodium hydroxide portion till precipitation occurred (at pH-6). It was filtered and residue was collected and dried. Residue was dissolved in 95% methanol. It was filtered and dried (10% yield). It was considered as an aqueous subfraction. Residue was collected (80% yield). Residue obtained was further dissolved twice in acetone through shaking and vortexing for 30 minutes. Filtrate was collected and dried at 45-50°C (20.0% yield). It was acetone subfraction. Residue was collected (50% yield). Residue obtained was further dissolved twice in chloroform through shaking and vortexing the solution for 30 minutes. Filtrate was collected and liquid-liquid extraction with sodium hydroxide was done in a separating funnel for purification of constitute and remove the sodium hydroxide soluble impurity. The lower layer was collected and dried at 45-50°C (32.0% yield). It was chloroform subfraction.
TLC plates were prepared using silica gel F. Ethyl acetate fraction, aqueous subfraction, acetone subfraction, chloroform subfraction, and residual subfraction were spotted on TLC plate. The solvent system was developed: chloroform (10): Benzene (70): Ethyl methyl ketone (10): Acetic acid (10). All these subfractions were run through this solvent system to separate chemical constituents found in the ethyl acetate fraction. Then TLC plate was observed under an UV chamber [Figure 1].
|Figure 1: Thin layer chromatography of various subfractions of leaves of Vitex negundo. EA = Ethyl acetate fraction, AQF = Aqueous subfraction, CF = Chloroform subfraction, AF = Acetone subfraction, RF = Residual subfraction|
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Chemical test for flavonoid
Shinoda test: Dissolve 0.5 g subfraction in 5 ml ethanol (95%). Add a few drops of HCl. Add magnesium turnings. The presence of pink color shows the presence of flavonoid.
| Methods|| |
Egg-albumin-induced asthma in guinea-pig 
This model was used to evaluate anti-eosinophilic activity and bronchial anti-inflammatory activity of various subfractions of leaves of Vitex negundo. Animals were divided into following groups.
The guinea pigs were sensitized with 1 ml of 10% egg-albumin intraperitoneally. After a week of sensitization with egg-albumin, all the animals were treated with respective treatments orally for a week. Two hours after the last dose of treatment (i.e., 15 th day of sensitization) the animals were to be tested with 0.5 ml of 2% egg-albumin intravenously through the saphenous vein. After 1 hour of challenge with egg-albumin, animals of all groups were examined for serum bicarbonate level by the method described in section 1.1. Three hours postchallenge, tracheo-bronchial tree was lavaged with saline to collect the bronchoalveolar lavage (BAL) to count total and differential WBC by the method described in section 1.2.
- Group 1 Normal group (vehicle control)
- Group 2 Egg-albumin sensitized
- Group 3 Dexamethasone (5 mg\kg)
- Group 4 Aqueous sub fraction (AQF) of leaves of Vitex negundo (200 mg/kg)
- Group 5 Acetone sub fraction (AF) of leaves of Vitex negundo (200 mg/kg)
- Group 6 Chloroform sub fraction (CF) of leaves of Vitex negundo (200 mg/kg).
| Measurement of the Serum Bicarbonate (HCO3-) Level|| |
About 2-3 ml of blood is collected after 1 hour of the challenge of egg-albumin. The serum is separated from blood by minimum exposure to air and it is stored in a sealed tube till the bicarbonate level was estimated.
Pipette 5 ml saline is collected in a conical flask. Add 0.1 ml of the serum and two drops of the indicator, phenol red. Mix well. This is considered as control and test is titrated till same color develops as in the case of control. Pipette 4.0 ml saline in another conical flask. Add 1 ml 0.01N hydrochloric acid (HCl). Mix well. Add 0.1 ml of serum. Mix well. Add 2 drops of indicator is added. Mix well. In above mixture, 0.01N sodium hydroxide is added till the pH changed to 7.35 or color changed from yellow to red (same as in the case of the control). Note the titration reading R ml. Calculation: Serum bicarbonate level m.Eq/ltr = (1 - R) × 100
Bal fluid study: Total and differential leucocytes count
After 3 hours of challenge with egg-albumin or just prior to death of animal, the tracheobronchial tree is lavaged with 10 ml of saline by inserting cannula and the bronchoalveolar lavage fluid is collected. Centrifugation is done at 4000 rpm for 5 min. and the pellet was resuspended in 0.5 ml saline. Total and differential leucocytes are counted by fully automated hematology analyzer Sysmex XS - 800i made in Japan.
Histopathology of lungs
The lobes of lungs were isolated and stored in 10% formalin. Samples were send for the histopathology study in "SPANDAN DIAGNOSTICS PATHOLOGY LABORATORY" in Rajkot. Briefly the procedure used included fixation of the tissue with formalin, embedding in paraffin blocks, sectioning with microtome (0.7 μ thickness) and finally staining by the Haemotoxylin and Eosin stain technique. 
Hemotoxylin stains nucleus light blue which turns red in the presence of acid. The cell differentiation is achieved by treating the tissue with acid solution the counter staining is performed by using eosin which imparts pink color to cytoplasm.
Purification of aqueous sub-fraction
Dissolve the aqueous subfraction with 50 ml water in beaker and transfer it in separating funnel for liquid-liquid extraction with dichloromethane. Shake the funnel for 15 minutes for removal of chlorophyll and other impurity in the dichloromethane layer. Collect the aqueous layer, dry it, and use for sample analysis. 
Identification of aqueous sub-fraction
Aqueous subfraction (30 mg) was dissolved in methanol for recording UV spectra. UV spectra of aqueous sub-fraction was recorded with UV spectrophotometer in B.K. Mody Government Pharmacy College, Rajkot. IR spectra of aqueous subfraction was recorded with SHIMADZU FT-IR 8400 Spectrometer in chemistry department of Saurashtra University, Rajkot. The sample was prepared by mixing 30 mg aqueous subfraction and Potassium bromide. Mass spectra of aqueous subfraction (30 mg) dissolved in methanol was recorded with Shimadzu EI-MASS Spectrometer QP 2010 in chemistry department of Saurashtra University, Rajkot. FNMR spectra of the aqueous subfraction (50 mg dissolved in deuteron methanol) was recorded with bruker 500 MHz NMR spectrometer in analytical department of Central Salt and Marine Chemical Research Institute (CSMCRI), Bhavnagar.
| Results|| |
Effect of various subfractions of leaves of Vitex negundo on serum bicarbonate level in Guinea pig
Intravenous administration of egg-albumin on the 15 th day after antigen challenge in guinea pigs produced significantly higher serum bicarbonate level (56.3333 ± 0.8819, P < 0.05) when compared with normal control group (30.6667 ± 0.8819, P < 0.05). Animals pretreated with aqueous subfraction of leaves of Vitex negundo showed significantly lesser serum bicarbonate level (31.6667 ± 1.4529, P < 0.05), when compared with untreated sensitized animals (56.3333 ± 0.8819, P < 0.05) [Figure 2].
|Figure 2: Effect of different subfractions of leaves of Vitex negundo on serum bicarbonate level* Signifi cantly different from control at P < 0.05. Each bar in the graph represents mean ± SEM of six observations|
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Effect of various subfractions of Vitex negundo leaves on differential leukocytes count in bronchial alveolar Lavage in egg-albumin sensitized Guinea pigs
Significantly higher eosinophils count was observed in untreated sensitized animals (29.50 ± 2.5074; P < 0.05), when compared with normal control group (6.50 ± 1.5044;P < 0.05). Animals pretreated with aqueous subfraction and dexamethasone showed significant lesser eosinophil count [(9.50 ± 1.5044; P < 0.05) and (7.50 ± 0.5014; P < 0.05) respectively], when compared with untreated sensitized animals (29.5 ± 2.5074; P < 0.05) [Figure 3].
|Figure 3: Effect of different subfractions of leaves of Vitex negundo on differential leucocytes count *#Signifi cantly different from control at P < 0.05. Each bar in the graph represents mean ± SEM of six observations|
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Histopathology: Effect of various subfractions of Vitex negundo leaves-treated lungs in egg-albumin-sensitized Guinea pigs
As shown in [Figure 4], histopathology of lungs in normal guinea pig showed normal airway, blood vessels, and bronchoalveolar space. Histopathology of lungs of sensitized guinea pigs showed congestion of alveolar septa. Interstitial space surrounding airway is characterized by a dense, mixed cellular infiltrate of inflammatory cells like eosinophils and neutrophils which is due to marked inflammation in lungs. Histopathology of lungs of animals treated with dexamethasone (5 mg/kg) shows normal airway, blood vessels, and bronchoalveolar space. Histopathology of lungs of animals treated with aqueous subfraction (200 mg/kg) shows normal airway, blood vessels, and bronchoalveolar space. Histopathology of lungs of animals treated with acetone subfraction (200 mg/kg) and chloroform subfraction (200 mg/kg) showed congestion of alveolar septa. Interstitial space surrounding airway is characterized by a dense, mixed cellular infiltrate of inflammatory cells like eosinophils and neutrophils which is due to marked inflammation in lungs.
|Figure 4: Effect of various subfractions of Vitex negundo leaves on lungs in guinea pigs|
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Results were analyzed statistically using one-way analysis of variance followed by Tukey's test. Data were considered statistically significant at P < 0.05.
Identification of aqueous subfraction by UV, FT-IR, EI-MASS and H1 NMR spectroscopy
UV spectra of the aqueous subfraction in methanol showed λmax at 252, 273, and 348 nm. EI-Mass spectra exhibited a molecular ion peak at m/z 388. Fragmentation occurs at m/z 373 (M-CH3), 345(M-CO.CH3), 165 (M- CO.CH3.C9H8O4). IR spectra revealed bands at 3406, 3126, 3105, 3036, 3016, 2976, 2904, 2821, 1604, 1581, 1346, 1161, 1120, 1082, 848, 810, 744, 721, and 660 cm -1 . H 1 NMR (500 MHz, deuteron methanol): δ H at 4.01(s), 3.98(s), 3.97(s), 3.86 (s), 3.66(s), 6.95 (d), 7.70 (d), and 6.49(s).
| Discussion|| |
The present study was undertaken to evaluate the antiasthmatic activity of various subfractions of leaves of Vitex negundo. Alternative medicine for the treatment of various diseases is getting more popular. Many medicinal plants provide relief of symptoms comparable to that of conventional medicinal agents.  Vitex negundo seems to be a promising plant for treatment of bronchial asthma because of its reported immunomodulatory and anti-inflammatory activity.  In the present study, we have evaluated antieosinophilic of aqueous subfraction, acetone subfraction, and chloroform subfraction of ethyl acetate fraction of leaves of Vitex negundo.
The presence of eosinophils in the airways of asthma patients was recognized years ago in histological preparation of lung tissue.  Intravenous administration of egg-albumin to guinea pigs after 14 days of antigen challenge resulted in selective pulmonary eosinophillia, a response that has been associated with airway hyper-reactivity. , The evidence of eosinophils involvement in inflammatory reactions within the asthmatic lung has been provided, by demonstration of an increased bronchial eosinophillia in patients exhibiting a late asthmatic response.  Eosinophils cause epithelial damage by releasing major basic protein  which may lead to increased airway reactivity, either by exposing sensory nerve endings  or by removing the protective effects of an epithelial derived relaxant factor.  In our study, we have used sensitized guinea pigs to demonstrate the effect of various subfractions of Vitex negundo leaves on eosinophils accumulation following antigen challenge. The guinea pig is well suited for such studies since airway hyper reactivity and eosinophillia can be readily demonstrated in this species. , As compared with unsensitized animals, a significant increase in eosinophils count was observed in untreated sensitized animals challenged with egg-albumin. Aqueous subfraction of Vitex negundo leaves give protection against the increase in eosinophils count. Intravenous administration of egg-albumin causes sudden rise in serum bicarbonate level. This is mainly due to the higher carbon dioxide tension in blood which is transported as bicarbonates. The aqueous subfraction was found to be effective in reducing the serum bicarbonate level.
The aqueous subfraction was obtained as a crystalline solid with creamish yellow color. It was soluble in water, methanol, and alkaline solution. Its melting point was 162-164°C. It gave a positive shinoda test that indicated that it may contain flavonoid. UV spectra of aqueous subfraction showed λmax at 252, 273, and 348 nm. So it suggested flavone skeleton for the aqueous subfraction as shown in [Figure 5]. 
Identification of the aqueous subfraction was done from the molecular ion peak and the fragmentation pattern of mass spectra. According to these mass spectra data it may be 5-hydroxy-3, 6, 7, 3',4'-pentamethoxy flavone as shown in [Figure 6]. , It was supported and confirmed by bands observed in IR spectra and signals recorded in H' NMR spectra.
|Figure 6: Chemical structure of 5-hydroxy-3,6,7,3',4'-pentamethoxy flavone|
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IR spectra revealed bands at 3406 (intermolecular --OH stretching vibration), 3126, 3105, 3036, 3016 (aromatic C-H stretching vibration), 2976, 2904, 2821(-CH3 stretching in -OCH3), 1604 (aromatic -C=O group), 1581 (aromatic -C=C- shows benzene ring skeletal), 1346(-C-OH deformation vibration), 1161(-C-O-C- stretching), 1120,1082 (-C-OH stretching vibration), 848, 810 (tri-substituted benzene ring), 744,721 (aromatic -C-H deformation vibration) and 660 (di-substituted benzene ring). ,
In the H 1 NMR spectrum, five singlets were present at 4.01, 3.98, 3.97, 3.86, 3.66 which indicated that five methoxy groups are present. In the H' NMR spectrum, signals at 6.95 (1H, doublet) and 7.70 (2 H, doublet) suggested a 3',4'-disubstituted B-ring as shown in [Figure 3]. A singlet at 6.49 was due to H at 8 position as shown in [Figure 6]. 
| Conclusion|| |
It can be concluded that aqueous subfraction of leaves of Vitex negundo possesses antieosinophilic activity. It reduces bronchial hyperresponsiveness and bronchial inflammation. As per UV, EI-Mass, FT-IR, and H 1 NMR spectra analyses, the structure of the aqueous subfraction was determined as 5-hydroxy-3, 6, 7, 3',4'-pentamethoxy flavone. It may be useful in the treatment of asthma and various inflammatory, allergic, and immunologic diseases.
| Acknowledgements|| |
The authors are thankful to K.B. Institute of Pharmaceutical Education and Research, Gandhinagar for partial financial supports and R.K. College of Pharmacy, Rajkot for providing instrumental facility for animal study for this research work.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]